Deproteinization of a sample by precipitation or solvent extraction typically precedes the determination of drugs, endogenous metabolites or other small molecules in physiological liquids by high-performance liquid chromatography. This pretreatment prevents the accumulation of proteins in the column that would likely lead to clogging and fast deterioration of the separation efficiency. However, such pretreatment is time consuming and does not always give reproducible and quantitative results. Therefore, different methods have been developed that allow the direct injection of these complex samples into a chromatograph. The simplest is solid phase extraction (SPE) in which the SPE column is disposed of after each injection. The precolumn technique utilizing two columns in tandem has significant advantages over SPE. Several hundred injections can be made on the precolumn. However, the precolumn technique requires an additional pump, a column switching device and computer control of events.
In parallel, stationary phases based on modified porous silica have been developed to allow the direct injection of complex matrices such as plasma, serum, saliva, and urine into a column for the determination of drugs and metabolites without any pre-treatment (D. J. Anderson, Anal. Chem., 65 (1993) 434R).
The majority of direct injection stationary phases yet described in the literature are based on silica that has been modified in order to prevent the contact of protein molecules with hydrophobic or charged functionalities attached to surface of the stationary phase (Pinkerton U.S. Pat. No. 4,544,485). In each case, the modification of the pores is uniform and affects all pores regardless of their size. Moreover, some of these silica based packings have limited working pH range, ionic strength range, or do not allow any high concentrations of organic solvent in the mobile phase during the reversed phase chromatography. The modifications may also impair diffusion of the low molecular weight compounds that have to be separated, and the slow mass transfer results in lower column efficiencies (about 20-36,000 plates/m) when compared to those of typical reversed phase silica columns.
Polymeric stationary phases have gained considerable popularity in HPLC due to their chemical stability in the entire pH range, broad variety of available surface groups chemistries and polarities. Styrene-divinylbenzene copolymers are the most often used polymeric stationary phases. Their highly hydrophobic surface accounts for their extensive use in reversed-phase chromatography and size-exclusion chromatography in non-aqueous media. A search for more hydrophilic stationary phases is still in progress in order to develop polymeric media for the separation of water soluble hydrophilic compounds and proteins without damaging their biological activity. Though more rugged in terms of chemical resistance, the polymeric stationary phases are essentially less efficient than silica based phases with efficiencies seldom exceeding 30,000 plates/m. Despite this limitation, the ability to modify the chemistry within specialized polymer phases remains a very significant advantage that frequently justifies their use. Recently, a few stationary phases for direct injection chromatography of complex samples based entirely on organic polymers were described in the literature (Hosoya et al., Chromatographia 38 (1994) 177; Beth et al. (Chromatographia 36 (1993) 351; Smigol et al, J. Liquid Chromatog. 17 (1994) 891).
The chromatographic method used for all of the separation media for direct injection chromatography is essentially the same. The sample that typically contains large protein molecules and small hydrophobic molecules (drugs, metabolites, etc.) is injected into the packed column and eluted with a mobile phase consisting of an aqueous buffer solution and an organic solvent. Due to the design of the separation media, the proteins are not retained within the column and elute in the void volume. In contrast, small more hydrophobic molecules are retained in the separation medium and elute after the proteins in an order that reflects their hydrophobicity. The typical drawback of such a direct injection separation technique is that the peak of the proteins typically has a long tail and less hydrophobic small molecules elute before the major peak reaches the baseline. This has an impact on the accuracy of the quantitative determination of small molecule drugs.
A far better approach would be to separate the small molecules first while the proteins remain completely retained on the top of the column and are released only by a different mobile phase at the end of the chromatographic run. Accordingly, it is an object of the present invention to achieve the complete separation of small molecules in complex samples using a technique that is inverse to the techniques of the prior art.